Project Summary. Compartmentalization of molecules into distinct volumes is essential for cellular life. Biomolecular condensates, composed of liquid-like, phase-separated protein and RNA, are important centers of compartmentalization in diverse contexts. Phase-separated structures also play central roles in pathological aggregates that cause disease. Despite the critical importance of phase separation in physiology and pathology, the regulatory mechanisms that govern when and where condensates form in cells are unknown. Our group discovered that biomolecular phase transitions play essential physiological roles in a multinucleate fungus (Zhang et al., Molecular Cell 2015; Langdon et al., Science 2018). Specifically, the RNA-binding protein Whi3 forms distinct, functional droplets with different RNA transcripts that regulate either the nuclear cycle or cell polarity. How do cells control assembly and patterning of different droplets in space and time? Recent reports demonstrated that membrane surfaces provide a powerful platform for promoting protein phase separation (Case et al., Science 2019; Huang et al., Science 2019). However, no studies have examined the role of membranes in controlling RNA-based phase transitions. In my preliminary studies, I found that Whi3 droplets stably associate with endomembranes in live cells. Moreover, I found that membranes promote phase separation of Whi3 in vitro at substantially lower concentration compared to free-diffusing protein in solution. These findings suggest that endomembrane surfaces regulate Whi3/RNA phase separation in space and time. Intriguingly, I also found that Whi3 partitions strongly to interfaces between contacting membranes, suggesting that regions of membrane contact between organelles or with the plasma membrane may regulate Whi3/RNA phase separation. How is Whi3 recruited to endomembranes? My preliminary findings reveal that an endomembrane-associated molecular chaperone component binds to Whi3 and tunes droplet properties. Importantly, molecular chaperones are known to influence droplet behavior, potentially defining the emergent identities and functions of droplets. Taken together, my findings suggest that (i) endomembranes promote and regulate protein/RNA phase transitions and (ii) membrane-associated chaperones control droplet properties to determine overall function. The objective of my proposed work is to elucidate the role of membranes and associated chaperones in regulating and patterning phase separation in space and time. The first specific aim will examine how membrane surfaces and interfaces control assembly of biomolecular condensates. The second specific aim will evaluate how membrane-associated chaperones regulate the emergent properties and functions of biomolecular condensates. This work will create innovative biophysical tools for the study of protein/RNA phase transitions in vitro and in live cells. The overall outcome of this research will be a deeper understanding of the key regulatory platforms that control phase separation. As such, my work will help reveal how cells build and maintain the fundamental compartments that control growth and division.